The integrin family of adhesion molecules are noncovalently-associated α/β heterodimers. To date, at least fourteen different integrin α subunits and eight different β subunits have been reported (Hynes, R O (1992) Cell 69:1-25). Lymphocyte function-associated antigen-1 (LFA-1) is a member of the leukocyte integrin subfamily. Members of the leukocyte integrin subfamily share the common β2 subunit (CD18) but have distinct α subunits, αL (CD11a), αM (CD11b), αX (CD11c) and ad for LFA-1, Mac-1, p150.95 and αd/β2, respectively (Springer, T A (1990) Nature 346:425-433; Larson, R S and Springer, T A (1990) Immunol Rev 114:181-217; Van der Vieren, M et al. (1995) Immunity 3:683-690). The leukocyte integrins mediate a wide range of adhesive interactions that are essential for normal immune and inflammatory responses.
Both integrin α and β subunits are type I transmembrane glycoproteins, each with a large extracellular domain, a single transmembrane region and a short cytoplasmic tail. Several structurally distinct domains have been identified or predicted in the α and β subunit extracellular domains.
The N-terminal region of the integrin α subunits contains seven repeats of about 60 amino acids each, and has been predicted to fold into a 7-bladed β-propeller domain (Springer, T A (1997) Proc Natl Acad Sci USA 94:65-72). The leukocyte integrin α subunits, the α1, α2, α10, α11, and αE subunits contain an inserted domain or I-domain of about 200 amino acids (Larson, R S et al. (1989) J Cell Biol 108:703-712; Takada, Y et al. (1989) EMBO J. 8:1361-1368; Briesewitz, R et al. (1993) J Biol Chem 268:2989-2996; Shaw, S K et al. (1994) J Biol Chem 269:6016-6025; Camper, L et al. (1998) J Biol Chem 273:20383-20389). The I-domain is predicted to be inserted between β-sheets 2 and 3 of the β-propeller domain. The three dimensional structure of the αM, αL, α1 and α2 I-domains has been solved and shows that it adopts the dinucleotide-binding fold with a unique divalent cation coordination site designated the metal ion-dependent adhesion site (MIDAS) (Lee, J-O, et al. (1995) Structure 3:1333-1340; Lee, J-O, et al. (199S) Cell 80:631-638; Qu, A and Leahy, D J (1995) Proc Natl Acad Sci USA 92:10277-10281; Qu, A and Leahy, D J (1996) Structure 4:931-942; Emsley, J et al. (1997) J Biol Chem 272:28512-28517; Baldwin, E T et al. (1998) Structure 6:923-935; Kallen, J et al. (1999) J Mol Biol 292:1-9). The C-terminal region of the αM subunit has been predicted to fold into a β-sandwich structure (Lu, C et al. (1998) J Biol Chem 273:15138-15147).
The integrin β subunits contain a conserved domain of about 250 amino acids in the N-terminal portion, and a cysteine-rich region in the C-terminal portion. The β conserved domain, or I-like domain, has been predicted to have an “I-domain-like” fold (Puzon-McLaughlin, W and Takada, Y (1996) J Biol Chem 271:20438-20443; Tuckwell, D S and Humphries, M J (1997) FEBS Lett 400: 297-303; Huang, C et al. (2000) J Biol Chem 275:21514-24). The C-terminal Cys-rich region of the β subunits appears to be important in the regulation of integrin function, as a number of activating antibodies to the β1, β2 and β3 subunits bind to this region (Petruzzelli, L et al. (1995) J Immunol 155:854-866; Robinson, M K et al. (1992) J Immunol 148:1080-1085; Faull, R J et al. (1996) J Biol Chem 271:25099-25106; Shih, D T et al. (1993) J Cell Biol 122:1361-1371; Du, X et al. (1993) J Biol Chem 268:23087-23092).
Electron microscopic images of integrins reveal that the N-terminal portions of the α and β subunits fold into a globular head that is connected to the membrane by two rod-like tails about 16 nm long corresponding to the C-terminal portions of the α and β extracellular domains (Nermut, M V et al. (1988), EMBO J 7:4093-4099; Weisel, J W et al. (1992) J Biol Chem 267:16637-16643; Wippler, J et al. (1994) J Biol Chem 269: 8754-8761).
LFA-1 is expressed on all leukocytes and is the receptor for three Ig superfamily members, intercellular adhesion molecule-1, -2 and -3) (Marlin, S D et al. (1987) Cell 51:813-819; Staunton, D E et al. (1989) Nature 339:61-64; de Fougerolles, et al. (1991) J Exp Med 1 74: 253-267). Substantial data indicate that the I-domain of LFA-1 is critical for interaction with ligands. Mutagenesis studies have shown that residues M140, E146, T175, L205, E241, T243, S245 and K263 in the I-domain are important for ligand binding (Huang, C et al. (1995) J Biol Chem 270:19008-19016; Edwards, C P et al. (1998) J Biol Chem 273:28937-28944). These residues are located on the surface) of the I-domain surrounding the Mg2+ ion, defining a ligand binding interface on the upper surface of the I-domain. The importance of the I-domain in ligand binding is further underscored by mAb blocking studies. A large number mAbs that inhibit LFA-1 interaction with its ligands map to the I-domain (Randi, A M et al. (1994) J Biol Chem 269:12395-12398; Champe, M et al. (1995) J Biol Chem 270:1388-1394; Huang, C et al. (1995) J Biol Chem 270:19008-19016; Edwards, C P et al. (1998) J Biol Chem 273:28937-28944). Two groups have recently shown that I-domain deleted LFA-1 lacks ligand recognition and binding ability, further demonstrating the role of the I-domain in LFA-1 function (Leitinger, B et al. (2000) Mol Biol Cell 11, 677-690; Yalamanchili, P et al. (2000) J Biol Chem 275:21877-82). The I-domains of other I-domain containing integrins have also been implicated in ligand binding (Diamond, M S (1993) J Cell Biol 120:545556; Michishita, M et al. (1993) Cell 72:857-867; Muchowski, P J et al. (1994) J Biol Chem 269:26419-26423; Zhou, L et al. (1994) J Biol Chem 269:17075-17079; Ueda, T et al. (1994) Proc Natl Acad Sci USA 91:10680-10684; Kamata, T et al. (1994) J Biol Chem 269:96599663; Kern, A et al. (1994) J Biol Chem 269:22811-22816).
Binding of LFA-1 to ICAMs requires LFA-1 activation. LFA-1 can be activated by signals from the cytoplasm, called “inside-out” signaling (Diamond, M S et al. (1994) Current Biology 4:506-517). Divalent cations Mn2+, Mg2+ and Ca2+ can directly modulate ligand-binding function of LFA-1 (Dransfield, I et al. (1989) EMBO J. 8:3759-3765; Dransfield, I et al. (1992). J Cell Biol 116:219-226; Stewart, M P et al. (1996) J Immunol 156:1810-1817). In addition, LFA-1 can be activated by certain mAbs that bind the extracellular domains of the αL or β2 subunit (Keizer, G D et al. (1988) J Immunol 140:1393-1400; Robinson, M K et al. (1992) J Immunol 148:1080-1085; Andrew, D et al. (1993) Eur J Immunol 23:2217-2222; Petruzzelli, L et al. (1995) J Immunol 155:854-866). The molecular mechanism for integrin activation is not yet well understood. It has been proposed that intramolecular conformational changes accompanying integrin activation increase integrin affinity for ligand, and this is supported by the existence of antibodies that only recognize activated integrins (Dransfield, I et al. (1989) EMBO J. 8:3759-3765; Diamond, M S et al. (1993) J Cell Biol 120: 545-556; Shattil, S J et al. (1985) J Biol Chem 260:11107-11114). One of such antibodies CBRLFA-1/5 binds to the Mac-1 I-domain very close to the ligand binding site (Oxvig, C et al. (1999) Proc Natl Acad Sci USA 96:2215-2220), providing further evidence that the I-domain itself undergoes conformational changes with activation.
Two different crystal forms of the Mac-1 I-domain have been obtained, and it has been hypothesized that the two structures represent the “active” and “inactive” conformation, respectively (Lee, J-O et al. (1995) Structure 3, 1333-1340; Lee, J-O et al. (1995) Cell 80:631-638). In the “active” form, crystallized with Mg2+, a glutamate from a neighboring I-domain provides the sixth metal coordination site, while in the “inactive” conformation, complexed with Mn2+, a water molecule completes the metal coordination sphere. The change in metal coordination is linked to a large shift of the C-terminal α-helix; in the putative “active” conformation, the C-terminal helix moves 10 Å down the body of the I-domain (Lee, J-O et al. (1995) Structure 3:1333-1340). Results from epitope mapping of mAb CBRM-1/5 that only recognizes activated Mac-1 have suggested that the conformational differences are physiologically (Oxvig, C et al. (1999) Proc Natl Acad Sci USA 96:2215-2220). The crystal and NMR structures of the LFA-1 I-domain have a conformation similar to the putative “inactive” conformation of the Mac-1 I-domain (Qu, A et al. (1995) Proc Natl Acad Sci USA 92:10277-10281; Qu, A (1996) Structure 4:, 931-942; Kallen, J et al. (1999) J Mol Biol 292:1-9; Legge, G B et al. (2000) J Mol Biol 295:1251-1264).
In addition to integrins, many pharmaceutically important proteins exist in two alternative three-dimensional structures, referred to as conformations or conformers. Often these proteins have important signaling functions, such as small G proteins, trimeric G protein α subunits, tyrosine kinases, and G protein-coupled receptors. Typically, one of these conformations and not the other is enzymatically active or has effector functions. Therefore, antibody or small molecule therapeutics that are specific for a protein in a particular conformation, for example, the active conformation, would have great advantages over non-selective alternatives.